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Pregnancy |
Imperial College Parturition Research Group, Wolfson and Weston Centre for Family Health, Institute of Reproductive and Developmental Biology, London W12 0HN, United Kingdom
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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gene regulation, parturition, progesterone, steroid hormones, steroid hormone receptors
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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Cytokines and prostaglandins act synergistically in synovial fibroblasts. Similarly, PGE2 and IL-8 act synergistically to remodel the cervix in rabbits [10, 11]. A major source of PGE2 is the amnion, which also contains high concentrations of the prostaglandin precursor, arachidonic acid. Prostaglandin synthesis in the amnion is entirely via inducible type 2 cyclooxygenase (COX-2) [12]. Expression of COX-2 increases exponentially with increasing gestational age to term. Expression of COX-2 mRNA increases 6-fold from early in the third trimester [13, 14] to term and shows a further doubling in association with labor onset [15]. There are parallel changes at the protein level. Both IL-1ß and IL-8 are principally produced in the chorion-decidua; however, there is also synthesis within the amnion. Expression of both IL-1ß and IL-8 increases in the amnion in the third trimester in parallel with expression of COX-2 [8]. IL-1ß concentrations also increase within the uterus with the onset of labor [16]. IL-1ß increases both IL-8 and prostaglandin synthesis [17, 18]. IL-1ß appears to act as an augmentor of the labor process. Progesterone is thought to contribute to myometrial quiescence during pregnancy through downregulation of a group of labor-associated genes, including IL-1ß, connexin 43, and MMP-9 [19–21]. Antiprogesterones are used clinically to terminate pregnancy in the second trimester [22] and have been shown to ripen the term human cervix [23].
The lower uterine segment has much in common with the cervix. The lower segment forms during the later stages of pregnancy and during labor, and although the upper segment contracts the lower segment relaxes and dilates. We therefore undertook a series of experiments to study the effect of progesterone on synthesis of IL-8 and PGE2 in lower segment fibroblast cells (LSF cells) obtained from the lower uterine segment at cesarean section. We examined the biochemical events in the lower segment of the uterus and the changes occurring in the LSF cells as a model for cervical fibroblasts. Our preliminary data revealed that, as expected, progesterone inhibits IL-8 synthesis in these LSF cells. Unexpectedly, we also found that progesterone inhibits COX-2 mRNA expression and PGE2 synthesis. We therefore extended our study to examine the effect of progesterone on IL-8 and COX-2 expression in the amnion, which is considered a major source of PGE2 and plays an important role in the onset of labor.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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Approval from the local ethics committee was obtained for this study. Informed consent was obtained from patients before the collection of tissue samples.
Cell Culture
Primary LSF cells were isolated from lower segment myometrial biopsies taken at elective caesarean section before labor. Tissue was chopped with a scalpel blade and then incubated for 2.5 h in Dispase (Gibco BRL, Gaithersburg, MD). The homogenate was then agitated with a sterile pipette tip in Dulbecco modified Eagle medium (DMEM; Sigma, St. Louis, MO) containing 10% heat-inactivated fetal calf serum (Helena Bioscience, Sunderland, U.K.), 1% L-glutamine (Gibco BRL), and 1% penicillin streptomycin (Gibco BRL) and then filtered through a single-cell filter. Cells were pelleted by centrifugation for 10 min at 1800 rpm and plated at a density of 1000 cells/ml. Cells were allowed to proliferate for 16–24 h until 80% confluence was reached, as determined by microscopic observation.
Primary amnion epithelial cultures were set up as previously described [24] and incubated in DMEM enriched with 10% batch-tested fetal calf serum (Sigma), 1% L-glutamine (Gibco BRL), and 1% penicillin streptomycin (Gibco BRL). Placentas were collected either at elective caesarean section before labor at term (38–41 wk) or after spontaneous vaginal delivery at term (38–41 wk). Primary amnion epithelial cells were plated at a density of 1000 cells/ml and allowed to achieve 80% confluence for 16–48 h. Cell culture experiments were set up to determine the effect of progesterone (1 µM–10 nM) on basal IL-8 and COX-2 mRNA activity and also the effect of progesterone on IL-1ß (1 ng/ml) treatment.
Transfection
Transient transfection was used to study promoter activity using 181 base pairs of the IL-8 promoter sequence (-135/+46) and a 2.2-kilobase COX-2 promoter sequence (-2375/+43) cloned into luciferase reporter vectors (PGL2 and PGL3, respectively; Promega, Madison, WI) [25, 26]. A cytomegalovirus-driven renilla expression vector was used to control for well-to-well transfection efficiency. Cells were plated and grown to 80% confluence, approximately 107 cells/well. Transfast (Promega) transfection agent was used to transfect 1 µg plasmid DNA per well (0.9 µg reporter and 0.1 µg renilla vector). Cells were incubated with transfection agent mastermix for 1 h and then flooded with DMEM. After 16 h, the medium was changed to serum-free medium. The medium was aspirated from the wells, and the plates were frozen at -20°C. Cells were lysed using Luclite reagent (Perkin Elmer Life Sciences, Zaverten, Belgium), and luciferase activity was measured with a Wallac Luminometer (Perkin Elmer). The luciferase reaction was quenched with 10 mM EDTA, and renilla activity was determined from the samples.
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ELISA |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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Reverse Transcription Polymerase Chain Reaction
Total RNA was extracted using RNA-stat solution. One microgram of RNA was treated with 2 units of DNase 1 (Ambion, Austin, TX) in 1x DNase buffer for 30 min. The DNase was denatured by heating to 75°C for 10 min in the presence of 5 mM EDTA to prevent thermal scission of the RNA, as suggested by the manufacturer. Reverse transcription (RT) was then performed at 37°C for 60 min in a final volume of 20 µl, with 0.2 µg of random hexanucleotide primers, Promega RT buffer with 1 unit of RNase inhibitor, 1 mM deoxynucleotide triphosphates, and 40 units of MMTV reverse transcriptase. A real-time quantitative polymerase chain reaction (PCR) was performed using Taqman. Primers for Taqman were designed using software from Applied Biosystems (Foster City, CA). Primers were generated by Hybaid, and the Taqman probe was purchased from Applied Biosystems. The cycling conditions were as follows: 94°C for 10 min and then 40 cycles of 95°C for 15 sec, 50°C for 15 sec, and 72°C for 10 sec.
Statistics
Luminometer data were analyzed and expressed as a proportion of the renilla control. Corrected luciferase values were then expressed as a percentage of the untreated control and analyzed by ANOVA with post hoc testing. ELISA readings were expressed as a proportion of each time point control and analyzed by ANOVA. Real-time PCR values for IL-8 and COX-2 mRNA were expressed as a proportion of glyceraldehyde phosphate dehydrogenase control values, and the same statistical analysis was applied.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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In LSF cells, progesterone had no effect on basal IL-8 or PGE2 release into the medium. IL-1ß increased IL-8 and PGE2 release into the medium, and only the highest concentration of progesterone (1 µM) repressed IL-8 activity or COX-2 release in this cell type (see Fig. 1, a and b). In amnion cells, progesterone had no effect on either unstimulated IL-8 or PGE2 synthesis (data not shown). In prelabor amnion cells, IL-1ß increased IL-8 synthesis 11-fold at 6 h of incubation. Coincubation with all concentrations of progesterone (1 µM–10 nM) significantly attenuated the effect of IL-1ß on IL-8 synthesis (Fig. 2a). In postlabor amnion cells, IL-1ß also increased IL-8 synthesis 10-fold. However, coincubation with progesterone did not attenuate the effect of IL-1ß on IL-8 synthesis at any dose (Fig. 2b). In prelabor amnion cells, IL-1ß increased PGE2 synthesis 5-fold by 6 h of treatment (Fig. 3a). Coincubation with progesterone only attenuated the effect of IL-1ß at the highest concentration (1 µM). For clarity, only the positive data are shown. In postlabor amnion cells, unstimulated PGE2 synthesis was 2-fold higher than that in prelabor cells (P = 0.009) (Fig. 3b). IL-1ß also increased PGE2 synthesis, but progesterone did not repress the effect of IL-1ß treatment (Fig. 3c).
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Messenger RNA data for both IL-8 and COX-2 paralleled that for IL-8 protein and PGE2 release. In LSF cells, IL-1ß increased both IL-8 and COX-2 mRNA expression, an effect that was significantly attenuated by progesterone (1 µM) (Fig. 4, a and b). In amnion cells, IL-1ß increased IL-8 mRNA expression, but this effect was attenuated by progesterone only in prelabor and not in postlabor cells (Fig. 5, a and b). COX-2 mRNA expression was increased by IL-1ß, and progesterone significantly repressed activity in prelabor but not postlabor amnion cells (Fig. 5, c and d). Although the effects of progesterone and IL-1ß were consistent, the proportional effects of either treatment varied among patients. The figures show a representative plot from one patient.
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Transfection experiments in LSF cells showed a pattern similar to that seen at the protein and mRNA levels. IL-1ß increased IL-8 activity, an effect that was significantly attenuated by progesterone (Fig. 6, a and b). The COX-2 promoter was not affected by IL-1ß or progesterone treatment. In prelabor amnion cells, IL-1ß increased IL-8 promoter activity by 250%, an effect that was significantly attenuated by progesterone (1 µM–10 nM) (Fig. 7a). In postlabor amnion cells, the effect of IL-1ß was lost, and progesterone did not affect promoter activity (Fig. 7b). In the amnion before labor, although IL-1ß did not increase COX-2 activity, activity of the COX-2 promoter was repressed to 60% of basal activity by progesterone alone at all concentrations. However, when IL-1ß was used only the highest concentration of progesterone repressed promoter activity to the same degree (Fig. 7c). After labor, the repressive effect of progesterone was lost (Fig. 7d).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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Whittle et al. [27] found that amnion epithelial and mesenchymal cells differ in the effect of glucocorticoids on prostaglandin synthesis and COX-2 expression. We studied amnion epithelial cells because they are a major source of prostaglandins. Amnion is easily obtained at cesarean section before labor and after vaginal birth and is therefore an accessible model for studying biochemical changes during parturition. The amnion epithelial cells were cytokeratin positive and vimentin negative (data not shown), suggesting that the amnion cells used in our experiments are of epithelial origin.
Much attention has been focused on the role of amnion-derived prostaglandins in cervical and lower segment ripening. Several research groups have demonstrated increased COX-2 expression and prostaglandin synthesis in amnion associated with labor [14, 28], and Van Meir et al. [29] found that prostaglandin dehydrogenase activity in fetal membranes decreases in the lower segment of the uterus in association with labor. Our findings suggest that direct production of both IL-8 and PGE2 by LSF cells may play a more important role in cervical and lower segment ripening than production by the amnion epithelium.
We found no differences in IL-8 production between amnion cells collected prelabor and those collected postlabor (data not shown). This finding is consistent with our previous results; although both IL-8 tissue content and release from chorion-decidua increase with labor, amnion IL-8 production is unchanged [8]. In the current studies, we found that PGE2 production was lower in prelabor than in postlabor amnion cells. In prelabor amnion cells, PGE2 production was stimulated by IL-1ß to levels similar to that found in postlabor amnion cells. This finding is consistent with previous data from our group showing that prostaglandin synthesis in fetal membranes collected prior to labor is variable. When low, synthesis can be stimulated with IL-1ß, but when high it cannot be stimulated. However, postlabor tissues have high levels of PGE2 synthesis that cannot be significantly further increased by IL-1ß [18]. The variation in levels of PGE2 synthesis in prelabor amnion cells probably depends on how close the patient is to the onset of clinical labor.
IL-8 expression is repressed by progesterone and dexamethasone in various cell types [3, 30–33]. The effect of dexamethasone on COX-2 has been widely studied [34–38]. Newton et al. [39] found that dexamethasone exerts its effect by influencing RNA stability. In almost all cell types except amnion epithelium [27], dexamethasone inhibits COX-2 expression [36, 39]. Our findings in human LSF cells are consistent with those of Ito et al. [7], who found that progesterone inhibited COX-2 expression and PGE2 production in rabbit cervical cells. However, there are few findings demonstrating the effect of progesterone with promoter studies.
The effect of progesterone upon cyclooxygenase has not been widely investigated. Progesterone treatment inhibits COX-2 mRNA in rat myometrium [40] and decreases PGE2 output from human first-trimester decidua [41]. In the current study, the effects of IL-1ß and progesterone on IL-8 and COX-2 in LSF cells were different. Progesterone affects the IL-8 but not the COX-2 promoter. The consistency of effect upon expression at the promoter, mRNA, and product levels suggests that IL-1ß mediates its effect upon IL-8 at the level of transcription and that, similarly, progesterone inhibits the effect of IL-1ß by inhibition of transcription. The COX-2 promoter is not influenced by IL-1ß treatment, suggesting its effect is posttranscriptional. Progesterone did not inhibit basal IL-8 or COX-2 expression, which suggests that the mechanisms of basal transcription are distinct from those stimulated by IL-1ß. The IL-8 promoter construct used in these studies contains binding sites for the transcription factors AP-1, C/EBP, NF-
B, and Oct-1 in close proximity but does not contain any consensus steroid response element. The COX-2 promoter used contains binding sites for the transcription factors AP-1, C/EBP, nuclear factor (NF)-B, and a possible glucocorticoid response element (GRE), but there is no progesterone response element or other steroid response element. Progesterone therefore appears to inhibit IL-1ß-stimulated IL-8 in LSF cells via a mechanism independent of binding to a steroid response element.
In amnion cells, we found the repressive effect of progesterone on IL-8 and COX-2 mRNA and on IL-8 protein and PGE2 release to be similar. There were also differences between prelabor and postlabor cells. The effect of IL-1ß and progesterone on IL-8 in prelabor cells was similar to that seen in LSF cells. However, in postlabor amnion cells, although IL-1ß stimulated IL-8 protein production this production was not supressed by progesterone. IL-1ß increases NF-B activity in amnion cells [42], and IL-8 expression depends upon NF-B [25]. Although there is abundant evidence for an effect of dexamethasone in inhibiting NF-B, information on the effect of progesterone is minimal. Miller and Hunt [43] showed that progesterone inhibits tumour necrosis factor 
expression in mouse macrophages through inhibition of NF-B, and King et al. [44] found that progesterone withdrawal is associated with increased NF-B activity in human endometrium. A mutual negative interaction between progesterone receptor (PR) and NF-B has been described in COS-1 and HeLa cells [45]. In the present study, we have shown that this interaction applies also to amnion cells. In association with labor, NF-B expression increases in amnion cells [42], whereas expression of PR decreases [46, 47]. The failure of progesterone to inhibit IL-8 in postlabor amnion cells may be simply through disappearance of PR. Glucocorticoid receptor (GR) has been demonstrated in human myometrium and trophoblast. Regulation of prostaglandin metabolism by glucocorticoids is antagonized by GR antagonists, demonstrating the importance of GR in the process of parturition. Repression of IL-8 within the amnion may be mediated via GR. Studies are in progress in our laboratory to investigate this possibility. We have seen only a 3-fold increase in IL-8 promoter activity stimulated by IL-1ß, whereas the mRNA and protein studies show much larger increases. This discrepancy may represent a limitation in the transient transfection approach. However, it also could reflect an effect of IL-1ß on IL-8 RNA stability. Our studies show that progesterone does inhibit IL-8 promoter activity, but if the effect of IL-1ß includes changes in IL-8 mRNA stability, then it appears that progesterone can also inhibit IL-8 mRNA stability. This possibility also requires further investigation.
In amnion cells, IL-1ß did not increase COX-2 promoter activity, again suggesting a posttranscriptional effect of IL-1ß. Progesterone repressed the COX-2 promoter but was only repressive at the highest concentration (1 µM) and during concurrent treatment with IL-1ß. IL-1ß increases NF-B activity in the amnion, which may account for the reduced potency of progesterone when IL-1ß is used concurrently. In previous studies, COX-2 activity increased through gestation and further increased with labor. Our studies included samples taken at cesarean section before labor. The timing of these deliveries has changed, and women are delivered in the 39th and 40th wk of gestation to avoid infant respiratory distress. This timing factor could account for the higher activity of the COX-2 promoter in amnion cells before labor. Thus, in amnion cells regulation of IL-8 and COX-2 does not occur through entirely common mechanisms. This conclusion is consistent with our earlier findings that although expression of both COX-2 and IL-8 in amnion cells increases at term prior to labor, only expression of COX-2 further increases with labor [8, 15].
We found an effect of progesterone on IL-1ß-stimulated IL-8 and COX-2 synthesis in prelabor amnion and LSF cells. We did not find that progesterone inhibited unstimulated endogenous expression of IL-8 or COX-2 at the level of mRNA or protein. IL-1ß concentrations within the uterus are elevated at the time of term and preterm labor [48]. IL-1ß production from amnion and chorion-decidua increases both in the third trimester of pregnancy and with the onset of labor [49]. In most species, the onset of labor is heralded by progesterone withdrawal. One of the actions of progesterone may be to inhibit IL-8 and COX-2 expression within the cervix and lower segment of the uterus, and when progesterone is withdrawn IL-1ß is able to potentiate the biochemical events that mediate cervical and lower segment ripening. Progesterone withdrawal does not occur in humans. However, several events do take place that might cause a functional withdrawal of progesterone. There is a decrease in PR expression in the amnion and a change in the ratio of PR expression within the chorion-decidua and myometrium from PR-B, which mediates the genomic effects of progesterone, to PR-A, which generally inhibits PR-B function [50, 51]. NF-B expression increases in the amnion and acts as a functional antiprogestin through inhibition of PR [42, 45].
Our findings may also have clinical significance. Progesterone inhibits IL-1ß-stimulated IL-8 synthesis in LSF cells and probably has a similar action in the cervix. Although the central role of progesterone in the maintenance of pregnancy has been recognized for several decades, there has been relatively little interest in the therapeutic use of progesterone to prevent preterm delivery. In a meta-analysis of 15 randomized control trials of progestational agents in pregnancy, Goldstein et al. [52] found that although progesterone therapy had no effect upon the rates of miscarriage, stillbirths, or neonatal death, it did reduce the risk of preterm delivery. Subsequently, Keirse [53] reported a meta-analysis of controlled trials of the prophylactic use of 17 alpha-hydroxyprogesterone caproate in women considered to be at high risk of miscarriage or preterm birth. In that study, 17 alpha-hydroxyprogesterone caproate did not protect against miscarriage but did appear to reduce the rate of preterm birth. Circulating progesterone concentrations are high during pregnancy and may be difficult to further increase pharmacologically. However, progesterone can be administered through the vagina directly into the cervix. Such prophylactic use of progesterone may be worthy of exploration.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Jenifer A.Z. Loudon, Imperial College Parturition Research Group, Wolfson and Weston Centre for Family Health, Institute of Reproductive and Developmental Biology, Hammersmith Hospital Site, Du Cane Road, East Acton, London W12 0HN, United Kingdom. FAX: 44 20 7594 2189; j.loudon{at}ic.ac.uk
Received: 19 December 2002.
First decision: 14 January 2003.
Accepted: 6 March 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSELISARESULTSDISCUSSIONREFERENCES |
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